Case report: Cheek acupuncture exhibits an immediate effect in relieving severe pain associated with nerve compression or damage of central nervous system and its potential mechanism of action.

Peripheral nerve compression or permanent damage of central nervous system (CNS) can trigger severe neuralgia to patients. Analgesic medicine or even surgery to remove nerve compression is commonly used for pain relief. But these treatments either are ineffective, have side-effect or can cause subsequent complications. Acupuncture, a technique that has been widely used in China and other Asian countries for thousands of years, is an alternative to relieve pain, although the mechanism of action is not fully understood. In this study, two patients who had symptoms of severe neuralgia associated with peripheral nerve compression or permanent damage/dysfunction of CNS and analgesic medicines are ineffective, underwent cheek acupuncture, a new technique established recent years by the author with the features of painless, standardization, simplicity, and precision. An immediate analgesic effect of the cheek acupuncture was observed without any side effects, and clinical remission was achieved after several sessions of treatments. It suggests that this new approach is an efficient alternative for pain relief induced by nerve impairment. The authors proposed a biological holographic model of triplet homunculi existing at the level of the local cheek, spinal cord, and cerebral cortex, to explain the immediate and accurate analgesic effect of the cheek acupuncture. These homunculi have the same structure, and synchronized sensations and actions that are mediated by afferent and efferent neurons, as the integrated human body. Therefore, the nociception and needling signals are sensed, transmitted, analyzed, and manipulated cooperatively and simultaneously among these homunculi with the subsequent pain relief in the body.

Pseudomonas aeruginosa Production of Hydrogen Cyanide Leads to Airborne Control of Staphylococcus aureus Growth in Biofilm and In Vivo Lung Environments.

Diverse bacterial volatile compounds alter bacterial stress responses and physiology, but their contribution to population dynamics in polymicrobial communities is not well known. In this study, we showed that airborne volatile hydrogen cyanide (HCN) produced by a wide range of Pseudomonas aeruginosa clinical strains leads to at-a-distance in vitro inhibition of the growth of a wide array of Staphylococcus aureus strains. We determined that low-oxygen environments not only enhance P. aeruginosa HCN production but also increase S. aureus sensitivity to HCN, which impacts P. aeruginosa-S. aureus competition in microaerobic in vitro mixed biofilms as well as in an in vitro cystic fibrosis lung sputum medium. Consistently, we demonstrated that production of HCN by P. aeruginosa controls S. aureus growth in a mouse model of airways coinfected by P. aeruginosa and S. aureus. Our study therefore demonstrates that P. aeruginosa HCN contributes to local and distant airborne competition against S. aureus and potentially other HCN-sensitive bacteria in contexts relevant to cystic fibrosis and other polymicrobial infectious diseases. IMPORTANCE Airborne volatile compounds produced by bacteria are often only considered attractive or repulsive scents, but they also directly contribute to bacterial physiology. Here, we showed that volatile hydrogen cyanide (HCN) released by a wide range of Pseudomonas aeruginosa strains controls Staphylococcus aureus growth in low-oxygen in vitro biofilms or aggregates and in vivo lung environments. These results are of pathophysiological relevance, since lungs of cystic fibrosis patients are known to present microaerobic areas and to be commonly associated with the presence of S. aureus and P. aeruginosa in polymicrobial communities. Our study therefore provides insights into how a bacterial volatile compound can contribute to the exclusion of S. aureus and other HCN-sensitive competitors from P. aeruginosa ecological niches. It opens new perspectives for the management or monitoring of P. aeruginosa infections in lower-lung airway infections and other polymicrobial disease contexts.

Primary ectocervical epithelial cells display lower permissivity to Chlamydia trachomatis than HeLa cells and a globally higher pro-inflammatory profile.

The tumoral origin and extensive passaging of HeLa cells, a most commonly used cervical epithelial cell line, raise concerns on their suitability to study the cell responses to infection. The present study was designed to isolate primary epithelial cells from human ectocervix explants and characterize their susceptibility to C. trachomatis infection. We achieved a high purity of isolation, assessed by the expression of E-cadherin and cytokeratin 14. The infectious progeny in these primary epithelial cells was lower than in HeLa cells. We showed that the difference in culture medium, and the addition of serum in HeLa cultures, accounted for a large part of these differences. However, all things considered the primary ectocervical epithelial cells remained less permissive than HeLa cells to C. trachomatis serovar L2 or D development. Finally, the basal level of transcription of genes coding for pro-inflammatory cytokines was globally higher in primary epithelial cells than in HeLa cells. Transcription of several pro-inflammatory genes was further induced by infection with C. trachomatis serovar L2 or serovar D. In conclusion, primary epithelial cells have a strong capacity to mount an inflammatory response to Chlamydia infection. Our simplified purification protocol from human explants should facilitate future studies to understand the contribution of this response to limiting the spread of the pathogen to the upper female genital tract.

FLCN-regulated miRNAs suppressed reparative response in cells and pulmonary lesions of Birt-Hogg-Dubé syndrome.

BACKGROUND: Birt-Hogg-Dubé Syndrome (BHDS) characterised by skin fibrofolliculomas, kidney tumour and pulmonary cysts/pneumothorax is caused by folliculin (FLCN) germline mutations. The pathology of both neoplasia and focused tissue loss of BHDS strongly features tissue-specific behaviour of the gene. Isolated cysts/pneumothorax is the most frequent atypical presentation of BHDS and often misdiagnosed as primary spontaneous pneumothorax (PSP). Deferential diagnosis of BHDS with isolated pulmonary presentation (PSP-BHD) from PSP is essential in lifelong surveillance for developing renal cell carcinoma. METHODS: The expression profiles of microRNAs (miRNAs) in cystic lesions of PSP-BHD and PSP were determined via microarray. The selected upregulated miRNAs were further confirmed in the plasma of an expanded cohort of PSP-BHD patients by reverse transcription quantitative PCR (RT-qPCR). Their diagnostic accuracy was evaluated. Moreover, the cellular functions and targeted signalling pathways of FLCN-regulated miRNAs were assessed in various cell lines and in the lesion tissue contexts. RESULTS: Cystic lesions of PSP-BHD and PSP showed different miRNAs profiles with a significant upregulation of miR-424-5p and let-7d-5p in PSP-BHD. The combination of the two effectively predicted BHDS patients. In vitro studies revealed a suppressive effect of FLCN on miR-424-5p and let-7d-5p expressions specifically in lung epithelial cells. The ectopic miRNAs triggered epithelial apoptosis and epithelial transition of mesenchymal cells and suppressed the reparative responses in cells and tissues with FLCN deficiency. CONCLUSION: The upregulation of miR-424-5p and let-7d-5p by FLCN deficiency occurred in epithelial cells and marked the PSP-BHD condition, which contributed to a focused degenerative pathology in the lung of PSP-BHD patients.

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

Puerarin prevents high-fat diet-induced obesity by enriching Akkermansia muciniphila in the gut microbiota of mice.

Growing evidence indicates that the gut microbiota plays a significant role in the pathophysiological processes of obesity and its related metabolic symptoms in the host. Puerarin, an active ingredient in the root of Pueraria lobate has been suggested to have a potent anti-obesity effect. Herein, we tested whether this effect of puerarin is associated with changes in the gut microbiota. In addition to reducing body weight, inflammation, and insulin resistance, puerarin administration significantly altered the composition of the gut microbiota. Notably, puerarin treatment greatly increased the abundance of Akkermansia muciniphila, a mucin-degrading bacterium known to be beneficial for host metabolism and significantly downregulated in high-fat diet-fed mice. Further experiments revealed that puerarin increased intestinal expression levels of Muc2 and Reg3g and protected intestinal barrier function (normal permeability) by increasing the expression of ZO-1 and occludin in vivo and in vitro. These data suggest that puerarin's enriching effect on A. muciniphila is mediated, at least in part, by a host cellular response to protect the host from diet-induced metabolic disorders and other diseases.

Rapid histology of laryngeal squamous cell carcinoma with deep-learning based stimulated Raman scattering microscopy.

Maximal resection of tumor while preserving the adjacent healthy tissue is particularly important for larynx surgery, hence precise and rapid intraoperative histology of laryngeal tissue is crucial for providing optimal surgical outcomes. We hypothesized that deep-learning based stimulated Raman scattering (SRS) microscopy could provide automated and accurate diagnosis of laryngeal squamous cell carcinoma on fresh, unprocessed surgical specimens without fixation, sectioning or staining. Methods: We first compared 80 pairs of adjacent frozen sections imaged with SRS and standard hematoxylin and eosin histology to evaluate their concordance. We then applied SRS imaging on fresh surgical tissues from 45 patients to reveal key diagnostic features, based on which we have constructed a deep learning based model to generate automated histologic results. 18,750 SRS fields of views were used to train and cross-validate our 34-layered residual convolutional neural network, which was used to classify 33 untrained fresh larynx surgical samples into normal and neoplasia. Furthermore, we simulated intraoperative evaluation of resection margins on totally removed larynxes. Results: We demonstrated near-perfect diagnostic concordance (Cohen's kappa, κ > 0.90) between SRS and standard histology as evaluated by three pathologists. And deep-learning based SRS correctly classified 33 independent surgical specimens with 100% accuracy. We also demonstrated that our method could identify tissue neoplasia at the simulated resection margins that appear grossly normal with naked eyes. Conclusion: Our results indicated that SRS histology integrated with deep learning algorithm provides potential for delivering rapid intraoperative diagnosis that could aid the surgical management of laryngeal cancer.

S1PR1 predicts patient survival and promotes chemotherapy drug resistance in gastric cancer cells through STAT3 constitutive activation.

BACKGROUND: S1PR1-STAT3 inter-regulatory loop was initially suggested to be oncogenic in several cancer cells. However, the clinical relevance of this mechanism in tumor progression, disease prognosis and drug response was not established. METHODS: The correlations between S1PR1 transcription, overall survival and chemotherapy response of GC patients were tested using a large clinical database. The relevance of S1PR1 expression and STAT3 activation in both tumor tissues and cancer cell lines was also tested. The effect of S1PR1 high expression achieved by persistent STAT3 activation on tumor cell drug resistance was investigated in vitro and in vivo. FINDINGS: An enhanced S1PR1 expression was highly related with a reduced overall survival time and a worse response to chemotherapy drug and closer correlation to STAT3 in gastric cancer patients. The issue chip analysis showed that the expressions of S1PR1 and STAT3 activation were increased in higher graded gastric cancer (GC) tissues. Cellular studies supported the notion that the high S1PR1 expression was responsible for drug resistance in GC cells through a molecular pattern derived by constitutive activation of STAT3. The disruption of S1PR1-STAT3 signaling significantly re-sensitized drug resistance in GC cells in vitro and in vivo. INTERPRETATION: S1PR1-STAT3 signaling may participate drug resistance in GC, thus could serve as a drug target to increase the efficacy of GC treatment. FUND: This work was supported by the National Natural Science Foundation of China (No. 81570775, 81471095), the grant from the research projects in traditional Chinese medicine industry of China (No. 201507004-2).

Immuno-modulatory functions of the type-3 secretion system and impacts on the pulmonary host defense: A role for ExoS of Pseudomonas aeruginosa in cystic fibrosis.

Number of previous reviews had described the structures and the various functions of the exotoxins produced by the type-3 secretion system of Pseudomonas aeruginosa and their roles in the interactions of this bacterium with host cells. In this review, we summarize some relevant data of literature on ExoS, an exotoxin from the type-3 secretion system of P. aeruginosa, with a particular focus on the role of this toxin in the airways innate response of the host to infection by this bacterium, and its implication in the elimination of Staphylococcus aureus from the airways of patients with cystic fibrosis.

High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling.

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Plasma microRNAs are associated with acute exacerbation in idiopathic pulmonary fibrosis.

BACKGROUND: Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) has high short-term mortality with unknown causes. To predict this malignant condition in clinics is challenging. In this study, we aim to demonstrate whether there are miRNAs that differ between AE-IPF and stable IPF, which may be served as reliable biomarker for AE-IPF prediction. METHODS: Human fibrotic-associated miRNAs arrays were designed to detect miRNAs expression in plasma of 3 AE-IPF patients, 3 Stable-IPF (S-IPF) patients and 3 normal controls (NC). Differentially expressed miRNAs between AE-IPF and S-IPF patients were selected for further analyses. The validation studies were carried out in plasma of 12 AE-IPF patients, 45 S-IPF patients and 51 healthy control subjects. Signaling pathways and cellular processes interacted with validated miRNAs were predicted by DIANA-miRPath. RESULTS: According to the array analysis, 6 miRNAs showed differentiated expression between AE-IPF and S-IPF patients (P < 0.05). In the validation studies, let-7d-5p was decreased in S-IPF and further decreased in AE-IPF, when compared to NC (0.0003 ± 0.0002 vs 0.003 ± 0.002, P < 0.01 and 0.0007 ± 0.0005 vs 0.003 ± 0.002, P < 0.01). While miR-25-3p was obviously decreased in S-IPF (0.0002 ± 0.0001 vs 0.0003 ± 0.0003, P < 0.01) but significantly increased in AE-IP (0.0023 ± 0.002 vs 0.0003 ± 0.0003, P < 0.01). In receiver-operator characteristic (ROC) curve analysis, the areas under the curve (AUCs) of miR-25-3p and let-7d-5p were 0.83 and 0.75, respectively. The sensitivity at fixed specificity of 90% was improved from 50% to 66.7% when the two miRNAs were combined. The functional prediction of miRNAs suggested that the loss of anti-fibrotic capacity and the gain of uncontrolled cell growth may be required in AE-IPF pathogenesis. CONCLUSIONS: In conclusion, miR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and S-IPF. A combination of these two miRNAs may be a potential biomarker for AE-IPF from IPF.

In vitro and in vivo evidence for an inflammatory role of the calcium channel TRPV4 in lung epithelium: Potential involvement in cystic fibrosis.

Cystic fibrosis (CF) is an inherited disease associated with chronic severe lung inflammation, leading to premature death. To develop innovative anti-inflammatory treatments, we need to characterize new cellular and molecular components contributing to the mechanisms of lung inflammation. Here, we focused on the potential role of "transient receptor potential vanilloid-4" (TRPV4), a nonselective calcium channel. We used both in vitro and in vivo approaches to demonstrate that TRPV4 expressed in airway epithelial cells triggers the secretion of major proinflammatory mediators such as chemokines and biologically active lipids, as well as a neutrophil recruitment in lung tissues. We characterized the contribution of cytosolic phospholipase A2, MAPKs, and NF-κB in TRPV4-dependent signaling. We also showed that 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, i.e., four natural lipid-based TRPV4 agonists, are present in expectorations of CF patients. Also, TRPV4-induced calcium mobilization and inflammatory responses were enhanced in cystic fibrosis transmembrane conductance regulator-deficient cellular and animal models, suggesting that TRPV4 is a promising target for the development of new anti-inflammatory treatments for diseases such as CF.

Does familial breast cancer and thymoma suggest a cancer syndrome? A family perspective.

Concurrence of breast cancer or thymoma with other malignancies in individual families is often observed, but the familial concurrence of breast cancer and thymoma has not yet been reported. Herein we reported a family encompassing five breast/ovarian cancer patients and two thymoma patients. Whole genome linkage analysis detected no haplotype co-segregating with both types of the tumors. In all patients with breast/ovarian cancer, genetic analysis revealed a clinically untested variant c.5141T>G in exon 18 of the BRCA1 gene, which could be a cancer-causing variant based on the functional study of Lee et al. (2010) and our current pedigree analysis. In the two thymoma patients in our family, targeted sequencing of RAD51L1 and BMP2 genes in and near the translocation site of chromosome 14 and 20 previously reported in two thymoma families, did not find any pathogenic mutation. In the present study, we identified a clinically unconfirmed BRCA1 variant segregating with breast/ovarian cancer patients in an individual family, suggesting it to be clinically functional. Our evidence, however, did not support the notion that the concurrent appearance of breast cancer and thymoma in our family represents a familial cancer syndrome caused by the same genetic disorder.

Staphylococcus aureus Adenosine Inhibits sPLA2-IIA-Mediated Host Killing in the Airways.

Staphylococcus aureus is a common cause of bacterial infections in respiratory diseases. It secretes molecules to dampen host immunity, and the recently identified adenosine is one of these molecules. The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host protein endowed with antibacterial properties, especially against Gram-positive bacteria such as S. aureus. However, the role of adenosine in sPLA2-IIA-mediated S. aureus killing by host is still unknown. The present studies showed that the S. aureus mutant lacking adenosine production (∆adsA strain) increased sPLA2-IIA expression in guinea pig airways and was cleared more efficiently, compared with the wild-type strain. S. aureus ∆adsA strain induced sPLA2-IIA expression by alveolar macrophages after phagocytic process via NOD2-NF-κB-dependent mechanism. However, S. aureus adenosine (wild-type and adsA-complemented strains) and exogenous adenosine downregulated S. aureus phagocytosis by alveolar macrophages, leading to inhibition of sPLA2-IIA expression. This occurred through inhibition of p38 phosphorylation via adenosine receptors A2a-, A2b-, and protein kinase A-dependent pathways. Taken together, our studies suggest that, in the airway, S. aureus escapes sPLA2-IIA-mediated killing through adenosine-mediated inhibition of phagocytosis and sPLA2-IIA expression.

iTRAQ-based quantitative proteomic analysis of cervical cancer.

Cervical cancer is the seventh most common cancer overall and the third among females. To obtain systematic insight into the protein profile that participates in cervical tumor oncogenesis and improve the current target therapies, iTRAQ labeling and NanoLC-MS/MS analysis were utilized to detect differentially expressed proteins in cervical cancer. As a result, 3,647 proteins were identified, among which the expression levels of 294 proteins in cervical cancer samples were distinct from the paired non-tumor samples. Further validation of the differentially expressed proteins, including G6PD, ALDH3A1, STAT1 and HSPB1, was carried out via qRT-PCR, western blot analysis and tissue microarray. Functional analysis of one of the highly expressed proteins, G6PD, was performed using RNA interference. Attenuated G6PD expression reduced the capacity of HeLa cells to migrate and invade in vitro. Our investigation complemented the understanding of cervical cancer progression. Furthermore, the present study supports the notion that suppressing the expression of G6PD may be a promising strategy in developing novel cancer therapeutic drugs.

Telomerase gene mutations and telomere length shortening in patients with idiopathic pulmonary fibrosis in a Chinese population.

BACKGROUND AND OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is an age-related disease and the most common manifestation of telomere-mediated disorders. METHODS: We collected detailed medical histories and DNA samples from 100 IPF patients seen at Nanjing Drum Tower Hospital Affiliated to Medical School of Nanjing University. All patients had sporadic IPF, with no family history reported. We screened all patients for telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) variants, and measured their telomere lengths in lymphocytes. RESULTS: Six novel telomerase gene mutations were identified in six IPF patients enrolled in the studies. They were two heterozygous mutations in TERC (257 G>A and 108 C>T) and four in TERT (R622H, T644M, V777L and F1032I). IPF patients with TERT/TERC mutations had significant thrombocytopaenia (160.167 ± 28.089 × 10(9)) compared with the non-mutation groups (191.018 ± 71.187 × 10(9), P = 0.047). All IPF patients with TERT/TERC mutations had shortened telomeres (0.656 ± 0.125) compared with the patients lacking TERT/TERC mutations (1.080 ± 0.6819, P = 0.0184). IPF patients lacking TERT or TERC mutations (1.080 ± 0.6819) had significantly shorter telomeres compared with age-matched healthy controls (1.244 ± 0.5890, P = 0.0355). CONCLUSIONS: Six novel mutations in the telomerase genes were identified for the first time in individuals diagnosed with sporadic IPF in a Chinese Han population. Shorter telomeres and mild thrombocytopaenia could be clues to association with telomerase gene mutation and sporadic IPF.

Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity.

Young cystic fibrosis (CF) patients' airways are mainly colonized by Staphylococcus aureus, while Pseudomonas aeruginosa predominates in adults. However, the mechanisms behind this infection switch are unclear. Here, we show that levels of type-IIA-secreted phospholipase A2 (sPLA2-IIA, a host enzyme with bactericidal activity) increase in expectorations of CF patients in an age-dependent manner. These levels are sufficient to kill S. aureus, with marginal effects on P. aeruginosa strains. P. aeruginosa laboratory strains and isolates from CF patients induce sPLA2-IIA expression in bronchial epithelial cells from CF patients (these cells are a major source of the enzyme). In an animal model of lung infection, P. aeruginosa induces sPLA2-IIA production that favours S. aureus killing. We suggest that sPLA2-IIA induction by P. aeruginosa contributes to S. aureus eradication in CF airways. Our results indicate that a bacterium can eradicate another bacterium by manipulating the host immunity.

Cytosolic phospholipase A2α enhances mouse mortality induced by Pseudomonas aeruginosa pulmonary infection via interleukin 6.

Pseudomonas aeruginosa pulmonary infection is a leading cause of death in numerous diseases such as cystic fibrosis (CF). The host cytosolic phospholipase A2α (cPLA2α) releases lipid mediators that play an important role in the pathogenesis of diseases, but its role in lung injury induced by P. aeruginosa infection is still obscure. Using an animal model of P. aeruginosa lung infection, we showed that the CHA strain of P. aeruginosa was more potent than the PAK strain in inducing mouse mortality and lung injury, and that both mouse mortality and lung injury were reduced in cPLA2α(-/-) mice as compared to cPLA2α(+/+) mice. This was accompanied by decreased levels of IL6 but not other inflammatory cytokines (IL1ß, KC and TNFα) in the bronchoalveolar lavage fluids (BALFs) of cPLA2α(-/-) mice. Given that CFTR(-/-) mice exhibit increased cPLA2α activation in the lung, the role of cPLA2α was further examined in this lung infection model. Compared to littermates, P. aeruginosa infection caused increased mortality in CFTR(-/-) mice with high IL6 levels in BALFs, which was attenuated by pharmacological inhibition of cPLA2α. In addition, compared to IL6(-/-) mice, an enhanced mortality was also observed in P. aeruginosa infected IL6(+/+) mice. Since alveolar macrophages (AMs) are the primary inflammatory cytokine source in the lung, murine AMs cell line (MH-S) were used to investigate the signalling pathways involved in this process. Incubation of MH-S cells with P. aeruginosa induced IL6 production, which was mediated by MAPKs ERK/p38 and was abolished by cPLA2α inhibitors. Furthermore, among cPLA2 downstream signalling pathways, only 15-lipoxygenase (15-LOX) and cyclooxygenase-2 (COX-2) were proven to participate in this P. aeruginosa-induced IL6 expression. Based on all these observations, we conclude that cPLA2α enhances P. aeruginosa-induced animal lethality in part via IL6 induction and that MAPKs ERK/p38, 15-LOX and COX-2 signalling pathways were involved in this process.

CFTR dysfunction induces vascular endothelial growth factor synthesis in airway epithelium.

Peribronchial angiogenesis may occur in cystic fibrosis and vascular endothelial growth factor (VEGF)-A regulates angiogenesis in airways. Peribronchial vascularity and VEGF-A expression were examined using immunocytochemistry and morphometric analysis in lung sections obtained in 10 cystic fibrosis patients at transplantation versus 10 control nonsmokers, and in two strains of Cftr-deficient mice versus wild-type littermates. Airway epithelial NCI-H292 cells and primary cultures of noncystic fibrosis human airway epithelial cells were treated with cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors (CFTR-inh(172) or PPQ-102) or transfected with a CFTR small interfering (si)RNA with or without a selective epidermal growth factor receptor tyrosine kinase inhibitor. Concentrations of VEGF-A and phosphorylated epidermal growth factor receptor were measured by ELISA. Peribronchial vascularity was increased in cystic fibrosis patients, but not in Cftr-deficient mice. VEGF-A immunostaining was localised to airway epithelium and was increased in cystic fibrosis patients and in Cftr-deficient mice. In cultured airway epithelial cells, treatment with CFTR inhibitors or transfection with CFTR siRNA induced a twofold increase in VEGF-A production. CFTR inhibitors triggered epidermal growth factor receptor phosphorylation that was required for VEGF-A synthesis. Cystic fibrosis airways at transplantation showed increased peribronchial vascularity and epithelial VEGF-A expression. CFTR dysfunction triggered epithelial synthesis of VEGF-A, which may contribute to vascular remodelling.

A novel bacterial resistance mechanism against human group IIA-secreted phospholipase A2: role of Streptococcus pyogenes sortase A.

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA(2)-IIA. Analysis of a set of clinical isolates demonstrated that an ~500-fold higher concentration of sPLA(2)-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA(2)-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA(2)-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (~30-fold) to sPLA(2)-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA(2)-IIA. Importantly, using transgenic mice expressing human sPLA(2)-IIA, we showed that the sortase A-mediated sPLA(2)-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA(2)-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA(2)-IIA and we showed that sPLA(2)-IIA contributes to the host defense against S. pyogenes infection.